FI82988B - FOERFARANDE FOER FRAMSTAELLNING AV MICROPROTEINER, SOM ERHAOLLITS UR CHLAMYDIA TRACHOMATIS, FOER IMMUNOLOGISKA BESTAEMNINGAR. - Google Patents

Description

1 82938

This invention relates to a method for the preparation of microbial proteins derived from Chlamydia trachoma-5 for immunoassay. That is, the invention relates to the use of detergents to dissolve a protein to elicit the antigenic properties of the proteins without eliminating the possibility of detecting the protein antigen in an immunoassay.

Detergents have long been known as reagents for dissolving many biochemical substances, especially microbial proteins and protein complexes. This possibility is useful for the release or detection of protein antigens, which can then be detected by immunoassay methods. However, the detergent in the antigen solution inhibits the immunoassay by preventing the antigen from binding to the antibody immobilized on the solid phase. To date, there has been no practical, inexpensive method available to prevent the detrimental effect of this detergent in an immunoassay. The fact that the sample is simply diluted is unsatisfactory. For example, if the immunoassay sample is diluted after dissolution so that the detergent concentration no longer interferes with the assay, a concomitant decrease in antigen concentration will result in an antigen level below the detection limit of the assay. Also, raising the initial concentration of antigen in the immunoassay sample to such an extent that the concentration of antigen in the sample. After 30 runs is above the limit of detection, it is often not practicable because the samples are often inevitably small. Thus, there is a need in the art for a method for dissolving proteins with a detergent that does not simultaneously inhibit immunoassays.

2,82988

A specific application for which detergents can be successfully used is the dissolution of the main outer membrane protein of Chlamyda trachomatis. This microorganism is another class of Chlamydiales in the family Chlamydiaceae out of two 5 species. Another species is Chlamydia psittaci. Approximately 15 different strains of Chlamydia trachomatis are etiologic agents for a number of human ocular and genital diseases, including trachoma, occlusive conjunctivitis, compartmental ligament run, "nonspecific" or nongonococcal urethritis, and rectal inflammation. C. trachomatis infection has spread throughout the human community. For example, it has been estimated that C. trachomatis is the cause of several million cases of nongonococcal urethritis each year.

15 Given the widespread nature of diseases caused by C. trachmatis, a reliable, simple and inexpensive test for the presence of this organism is highly desirable in order to provide appropriate treatment. The only serological test currently in use is the 20 microimmunofluorescence test. However, this test requires the use of C. trachomatis strains as a serological test antigen. In addition, the capacity to perform this test is limited to a limited number of laboratories worldwide. The test is very laborious, time consuming and difficult to pass.

This invention relates to a method for preparing a sample for immunoassay and is particularly useful for preparing microbial cell protein samples, such as Chlamydia trachomatis main membrane main protein samples. This particular protein has been found to be a species-specific antigen and thus useful in an immunoassay to detect the presence of this organism in an infected individual.

Before so-35 lupoproteins from organisms such as C. trachomatis can be used in an immunoassay, the cell wall containing 3,82988 proteins must be altered so that the protein is recognized by the appropriate antibody. Deter gentle treatment has been found to be effective for this purpose. Particularly valuable in dissolving cell wall protein are the lauryl sulfate salts of a nonionic detergent. To prevent interference with the detergent for subsequent immunoassay, an alkali or alkaline earth metal ion is included in the detergent solution in the method of the present invention. The detergent is soluble at high temperatures in the presence of such ions. At lower temperatures, such as room temperature, the detergent precipitates and can possibly be removed before performing an immunoassay.

The method of the invention is characterized by mixing a microbial protein sample from Chlamydia trachomatik in an aqueous solution containing a protein-soluble amount of an ionic detergent having an alkali or alkaline earth metal ion as a cation to obtain a sample solution; incubating the sample at an elevated temperature for a sufficient time to dissolve the protein; and cooling the sample solution in the presence of a compound having an alkali or alkaline earth metal ion as a cation, which compound acts as a temperature-dependent precipitating compound, thus causing the detergent to precipitate from solution at or slightly above room temperature, but does not affect the solubility of the detergent. temperatures, the cation of the compound being different from the cation of the detergent.

The method of the invention uses a temperature-dependent precipitant to remove a protein-precipitating detergent from the immunoassay sample. Detergents are very effective protein solubilizers, especially for microbial cell proteins such as the main outer membrane protein of Chlamydia trachomatis. However, since detergents interfere with the immunoassay by inhibiting antigen binding, they must be removed from the sample solution before testing.

It has been found that when a compound containing an alkali or alkaline earth metal ion is included in the sample solution, the solubility of a detergent such as lauryl sulfate in Liu-5 in water is greatly reduced at or slightly above room temperature (i.e., about 30 ° C or below). , but the effect on solubility is small or non-existent at elevated temperatures. Because these compounds have the ability to cause temperature-dependent precipitation of the detergent, they are sometimes referred to hereinafter as precipitating compounds. It is to be understood that this does not mean that the compounds themselves compound the detergent, but that they only cause precipitation. Alkali and alkaline earth metals refer to elements of groups I and II of the Periodic Table of the Elements, including but not limited to lithium, sodium, potassium, magnesium, calcium and barium. Thus, for example, an aqueous solution of sodium or lithium lauryl sulfate and potassium ion can be used to dissolve the microbial protein at an elevated temperature. The solution 20 is then cooled to approximately room temperature or slightly above, which causes the detergent to precipitate. Optionally, the detergent can be removed by centrifugation or filtration. Whether or not the precipitated detergent is removed, an immunoassay is then performed on the protein-nose sample without interfering with the detergent. Since the doping compound has no part in the invention before the cooling step, it can be added to the solution after dissolving the protein instead of initially combining it with the detergent.

Suitable detergents for use in the present invention include lauryl sulfates such as sodium dodecyl sulfate and lithium dodecyl sulfate. The detergent is generally present in the solution at a concentration of about 0.01 to 2.0% (w / v). The concentration is preferably about 0.01 to 1.0% (w / v), preferably about 0.05 to 5,82988 0.1% (w / v).

The precipitating compound is present in the solution in an amount of about 0.01 to 2.0 mol / l. The concentration is preferably about 0.01 to 1.0 mol / l, preferably about 0.05 to 1.0 mol / l. Any water-soluble compound having an alkali or alkaline earth metal ion as a cation can be used. The compound may be, for example, a phosphate, chloride or carbonate salt. The choice of doping compound to be used is influenced by the detergent used. For example, sodium dodecyl sulphate precipitates best at room temperature under the influence of sufficient potassium, calcium or barium ion contents, while lithium dodecyl sulphate precipitates sufficiently well under the influence of magnesium and sodium ions in addition to the above-mentioned ions.

An aqueous solution of the detergent and precipitating compound is added to a sample, such as a cervical or urethral sample. The sample is thoroughly mixed with an aqueous solution of the detergent and the precipitating compound to obtain a sample solution. When the sample solution is stirred at room temperature, the detergent remains insoluble. The solution in which the detergent is insoluble is heated from room temperature to about 60-120 ° C and maintained at this temperature for about 5-30 minutes. At this incubation temperature, the detergent dissolves and dissolves the microbial protein, exposing the antigen for immunoassay.

After incubation, the solution is rapidly cooled using an ice bath or other suitable means to a sufficiently low temperature to precipitate the detergent. Alternatively, the solution may be cooled slowly until it reaches room temperature. A suitable buffer, such as phosphate buffer (e.g., a solution containing 0.2 mol / L sodium phosphate and 0.1% (w / v) bovine serum albumin, pH 7.4), may be added before or after incubation. The buffer maintains the pH of the solution in the range of about 6.5 to 8.0. After cooling, the precipitated 6,82933 detergent can be removed or left in solution. If the detergent is left in solution, it has little or no effect on the immunoassay because it is not in solution.

As mentioned above, a particular use of the process of this invention is the solution of Chlamydia trachomatis. The major outer membrane protein of C. trachomatis accounts for about 60% of the total protein in the outer membrane of the microorganism and its size, i.e., the subunit 10, has a molecular weight of 38,000 to 44,000 daltons with an average molecular weight of 39,500 daltons. Henceforth, for ease of reference, this group of C. trachomatis outer membrane main proteins is abbreviated MP 39.5, which comes from the term "outer membrane main protein having a molecular weight of 39,500 daltons in its subunit 15". MP 39.5 is a species-specific antigen in the sense that, when tested against antibodies to all C. trachomatis serotypes, it reacts species-specifically. As an antigen, MP 39.5 provides a basis for the identification of all C. trachomatis serotypes.

Species-specific antibodies against an antigen such as MP 39.5 can be generated by appropriate inoculation procedures in laboratory animals such as mice and / or rabbits. Antibodies developed in animals can be used to study infection in other mammals.

These assays can be performed using well-known procedures used to examine the presence of bacterial antigen in an infected subject. Once the producer of monospecific antibodies has been confirmed in antigen-inoculated experimental animals, samples obtained from mammals suspected of being infected can be examined by either direct or indirect testing procedures. Assay methods such as enzyme-linked immunosorbent assay or radioimmunoassay are suitable for these purposes.

i 7 82988

In a direct study, an anti-antigen mono-specific antibody is covalently or non-covalently attached to a solid phase carrier system. As is customary in such methods, the carrier-5 system may be glass, plastic or the like. The solid phase support and the monospecific antibody attached thereto can be incubated with a sample previously prepared from an infected individual prepared in accordance with the invention.

The monospecific antibody pre-labeled with the radionuclide or coupled to the enzyme by known methods is then equilibrated against the carrier system. Any antigen present in the sample, which is bound to the antibody on the carrier system, in turn binds to the radionuclide-labeled or enzyme-linked antibody. If a radionuclide-labeled antibody is used, the residual radioactivity of the sample can then be determined. When an antibody coupled to the enzyme is used, a substrate specific for the enzyme is added to the reaction mixture containing the solid support, and the resulting color change is recorded spectrophotometrically. This color change is compared to the color change caused by samples known to be free of antigen. The presence of an antigen such as MP 39.5 in mammalian displays can thus be directly investigated.

Alternatively, indirect research methods may be used. More specifically, the antigen may be covalently or non-covalently bound to a suitable solid phase carrier system. A sample from 30 individuals suspected of being infected is prepared as described above. The sample is then mixed with a known amount of a radionuclide-labeled or enzyme-linked antibody to this antigen that has been previously isolated from a laboratory animal. The sample antibody mixture can then be incubated with the solid phase carrier system and its associated β 82988 antigen.

The radioactivity of the solid support system or the color change of the system attached to the enzyme is measured and compared to standard samples treated in the same manner and free of antigen.

The ability of a clinical sample suspected of containing a particular microorganism to inhibit the binding of radionuclide-labeled or enzyme-linked antibodies to a solid support indicates the presence or absence of antigen in the sample. Any blockage is an indication of infection.

Those familiar with such research methods know other suitable research methods.

Example 1 illustrates the method of this invention when used in conjunction with C. trachomatis MP 39.5. Table I compares the reference immunoassay to an assay using a sample prepared according to the invention.

Example 1 To 20 450 μl of Chlamydia trachomatis stock solution in phosphate buffer (0.2 mol / l sodium phosphate and 0.1% (w / v) bovine serum albumin, pH 7.4), a sufficient amount of potassium chloride was added to the sample. to adjust the potassium ion content to 0.1 mol / l. Approximately 50 μΐ of a solution containing 1% by weight of SDS (sodium dodecyl sulfate) in water was then added and the sample was heated to 100 ° C. The sample was incubated at this temperature for 10 minutes and then cooled to room temperature in an ice bath to precipitate SDS. Immunoassays were then performed on stock solutions diluted 1: 1, 1: 2, 1: 4, 1: 8 and 1:16 without removing the precipitated detergent. The control sample was otherwise prepared in the same manner, but no addition of potassium chloride was omitted. The results presented in Table 1 show that a positive result for Chlamydia 3.5 is achievable with much dilute

II

9,82988 solutions than the control sample to which no precipitant was added. This indicates a significant improvement in sensitivity.

The best mode and preferred embodiments are described herein. Modifications and alterations may also be made to the invention.

Table I

Chlamydia strain EIA results2 solution1 la- According to the example- Untreated 10 melt ratio treated (reference sample) 1:16 0.53 0 1: 8 0.94 0 1: 4 1.31 0.11 1: 2 1.65 0.27 15 Undiluted 1.81 0.45 1 in undiluted solution 5 x 107 bases / ml in 0.02 M sodium phosphate buffer 2 expressed as S / N of the absorbance of the absorbance measured at 450 nm.

Claims (24)

A process for producing microb proteins obtained from Chlamydia trachomatis, for immunological determinations, characterized in that a microb protein sample obtained from Chlamydia trachomatis is mixed with an aqueous solution of protein-dissolving quantity of an ionic detergent having a alkali or an alkaline earth metal ion as a cation, to obtain a sample solution; incubating the sample at an elevated temperature long enough to dissolve the protein; and cooling the sample solution in the presence of a compound having an alkali or an alkaline earth metal ion as a cation, which compound acts as a temperature dependent precipitating compound which thus causes the detergent to precipitate out of the solution at room temperature or slightly above, but does not affect the solubility of the detergent. at elevated temperatures, the cation of the compound being different from the cation of the detergent. Process according to claim 1, characterized in that the detergent is present in the sample solution at a concentration of approx. 0.01 - 2.0% (w / v). 25 3. A process according to claim 2, characterized in that the precipitating compound is present in the solution at a concentration of approx. 0.01 - 2.0 mol / l. 4. A process according to claim 3, characterized in that the precipitating compound contains a cation which is a sodium, potassium, calcium, barium or magnesium ion. Process according to claim 4, characterized in that the sample solution is incubated at a temperature ρέ approx. 60 - 120 ° C approx. 5 - 30 minutes. 14 82938 6. A process according to claim 5, characterized in that the detergent is present in the sample solution at a concentration ρέ approx. 0.01-1.0% (w / v). 5 7. A process according to claim 6, characterized in that the compound is present in a solution at a concentration of approx. 0.01 - 1.0 mol / l. 8. Process according to claim 7, characterized in that the solution contains a buffer 10 for pouring the pH of the solution at a value of approx. 6.5 -8.0. 9. A process according to claim 8, characterized in that the detergent is lauryl sulfate. 10. A process according to claim 9, characterized in that the precipitating solution is a water-soluble site. 11. Process according to claim 10, characterized in that the detergent is sodium dodecyl sulfate and the precipitated solution contains a cation or is a potassium, calcium or barium ion. Method according to claim 11, characterized in that the solution is incubated at a temperature of approx. 100 ° C for approx. 10 minutes. 13. Process according to claim 12, characterized in that the precipitating compound is potassium chloride and it is present in the solution at a concentration of approx. 0.05 - 1.0 mol / l. 14. A process according to claim 13, characterized in that the detergent is present at a concentration of approx. 0.05 - 0.1% (w / v). 15. A process according to claim 10, characterized in that the detergent is lithium dodecyl sulfate. Method according to Claim 15, characterized in that the solution is incubated at a temperature of 82988 per cent. 100 eC for approx. 10 minutes. 17. A process according to claim 16, characterized in that the precipitating compound is potassium chloride and it is present in the sample solution at a concentration of approx. 0.05 -1 mol / l. 18. A process according to claim 17, characterized in that the detergent is present at a concentration of approx. 0.05 - 0.1% (w / v). 19. A method according to claim 14, characterized in that the test solution is examined by radioimmunological determination. 20. A method according to claim 18, characterized in that the solution is examined by radioimmunological determination. 15 21. A process according to claim 19, characterized in that the precipitating compound is added to the test solution before the heating stage. Method according to claim 19, characterized in that the precipitated compound is added to the sample solution after the incubation stage. 23. A process according to claim 20, characterized in that the precipitating compound is added to the test solution before the heating stage. Process according to claim 20, characterized in that the precipitated compound is added to the sample solution after the incubation stage.